Compositions, methods and uses for inducing viral growth

ABSTRACT

Embodiments herein report methods, compositions and uses for inducing and/or accelerating viral growth. In certain embodiments, methods, compositions and uses generally related to copolymer compositions for inducing viral growth, reducing lag time and/or increasing viral plaque size. In other embodiments, methods, compositions and uses of copolymer compositions can be for inducing flaviviral growth, reducing lag in growth and/or increasing plaque size.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/120,262, filed on Dec. 5, 2008, and theprovisional application is incorporated herein by reference in itsentirety for all purposes.

FEDERALLY FUNDED RESEARCH

The studies disclosed herein were supported in part by grant number5U01AI07443 from the National Institutes of Health. The U.S. Governmentmay have certain rights to practice the subject invention.

FIELD

Embodiments of this application generally report methods, compositionsand uses for accelerated or enhanced viral growth. In certainembodiments, this application reports methods, compositions and uses ofcopolymer compositions for inducing accelerated viral growth and/orincreasing viral plaque size. In other embodiments, methods,compositions and uses of copolymer compositions are reported foraccelerating flaviviral growth, reducing flaviviral lag time and/orincreasing flaviviral plaque size.

BACKGROUND

Vaccines to protect against infectious diseases have been used toimprove human and animal health. One successful technology for viralvaccines is to immunize animals or humans with a weakened or attenuatedstrain of the virus (a “live, attenuated virus”). Due to limitedreplication after immunization, the attenuated strain does not causedisease. However, the limited viral replication is sufficient to expressthe full repertoire of viral antigens and generates potent andlong-lasting immune responses to the virus. Thus, upon subsequentexposure to a pathogenic strain of the virus, the immunized individualis protected from disease.

Recent technical advances, such as reassortment, reverse genetics andcold adaptation, have led to advances of live, attenuated viruses forinfluenza and rotavirus. A number of live, viral vaccines developed withrecombinant DNA technologies are in human clinical testing, includingvaccines for West Nile disease, dengue fever, malaria, tuberculosis andHIV. These recombinant viral vaccines rely on manipulation ofwell-characterized attenuated viral vaccines, such as adenovirus,vaccinia virus, yellow fever 17D or the dengue virus, DEN-2 PDK-53. As agroup, live attenuated viral vaccines are amongst the most successfulmedical interventions in human history, second only to the advent ofantibiotics and hold the promise to improve public health throughout theworld.

Other vaccines have been developed by inactivating viruses after growthin cell culture. These “killed virus” vaccines induce immune responsesdue to the presence of high concentrations of antigen present. Examplesof effective killed viral vaccines include, but are not limited to,vaccine for rabies, influenza, hepatitis A, and poliovirus.

Flaviviruses cause a number of human and animal diseases of significantimpact. They are enveloped viruses with a RNA genome of approximately11,000 bases. Most of the flaviviruses are transmitted by an arthropodvector, commonly mosquitoes. There are over 70 different flavivirusesthat are grouped into three major categories based on serology: thedengue group, the Japanese encephalitis group and the yellow fevergroup. Expanding urbanization, worldwide travel and environmentalchanges (such as deforestation or rain patterns) have lead to theemergence of several flaviviruses as threats to human public health.Such viruses include, but are not limited to, yellow fever virus, thedengue viruses, West Nile virus, Japanese encephalitis virus, andtick-borne encephalitis viruses.

Both live, attenuated viral vaccines and killed virus vaccines have beendeveloped that are safe and protect against flavivirus diseases, forexample, yellow fever and Japanese encephalitis.

SUMMARY

Embodiments of this application generally relate to methods,compositions and uses for inducing, enhancing and accelerating viralgrowth. In certain embodiments, this application reports methods,compositions and uses of copolymer compositions for inducing acceleratedviral growth and/or increasing viral plaque size. In other embodiments,methods, compositions and uses of copolymer compositions are reportedfor accelerating flaviviral growth, reducing flaviviral lag time and/orincreasing flaviviral plaque size.

One limitation for producing vaccines has been large-scale manufactureand in vitro growth of the viruses to support the demand of vaccines.Thus, one of the needs that exist in the art is for enhancing andaccelerating viral growth. Certain embodiments of the present inventionconcern methods and compositions for enhancing and accelerating viralgrowth. These compositions are of use, for example, in production ofviral vaccines and viral byproducts of use in other technologies such asmanufacturing of viral-related gene therapies and other viral products.In addition, embodiments herein may be of use to enhance or accelerategrowth of viral cultures of use in killed virus vaccines.

Certain compositions disclosed herein can include copolymers alone or incombination with other agents or compounds for enhancing andaccelerating viral growth. Other embodiments herein concern combinationsof excipients that enhance growth of live attenuated viruses. Copolymersof use herein include, but are not limited to, Pluronic F127, PluronicF68, Pluronic P123, Pluronic P85, other polyethylene oxide-polypropyleneoxide (EO-PO) block copolymers of greater than 3,000-4,000 MW orcombinations thereof.

In accordance with these embodiments, viruses can include, but are notlimited to, Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus,Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus,Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus,Parvovirus, Papovavirus, Adenovirus, Herpes virus, and Poxvirus. Someembodiments, directed to compositions of use in viral cultures, caninclude, but are not limited to, cultures having one or more viruses,such as a mixture of viral species or a single species, or one or morelive, attenuated viruses grown in one or more copolymer compositionsalone, or in combination with other agents.

In other embodiments, compositions contemplated herein can increaseplaque size in a reduced or similar time period of growth, compared to acontrol culture without the disclosed composition, for use in tittering,manufacturing or measuring the activity of virus preparations. In someaspects of the present invention, higher viral titers may be obtained inreduced time periods. Alternatively, compositions contemplated hereincan reduce lag time or accelerate growth time up to several dayscompared to control viral cultures not using compositions contemplatedherein.

Other embodiments concern virus populations for use in formulations andmethods directed to vaccine formulations capable of reducing orpreventing onset of a medical condition caused by one or more of theviruses contemplated herein. In accordance with these embodiments,medical conditions may include, but are not limited to conditions and/orinfections including West Nile, dengue fever, Japanese encephalitis,Kyasanur forest disease, Murray valley encephalitis in Australia and NewGuinea, Kunjin virus (a relative of West Nile), Alkhurma hemorrhagicfever, St. Louis encephalitis, hepatitis C virus infection, tick-borneencephalitis, yellow fever, the Usutu, Koutango, Yaonde viruses inAfrica, and Cacipacore in South America. In certain embodiments,production time for generating vaccine formulations may be reduced byusing compositions contemplated herein for accelerating viral growthproduction and manufacture, reducing lag time and/or increasing plaguesize of viral populations.

In certain embodiments, viral cultures contemplated for productionherein may be used in compositions including, but not limited to,partially or wholly dehydrated or hydrated vaccine formulations or otherviral formulations.

In certain embodiments, a live attenuated virus for use in a vaccinecomposition contemplated herein may include, but is not limited to, oneor more live, attenuated flavivirus vaccines, including but not limitedto, attenuated yellow fever viruses (e.g. 17D), attenuated Japaneseencephalitis viruses, (e.g. SA 14-14-2), attenuated dengue viruses (e.g.DEN-2/PDK-53 or DEN-4Δ30), attenuated chimeric West Nile vaccines, orrecombinant chimeric flaviviruses.

Other embodiments concern kits for growing viral cultures contemplatedherein. It is contemplated that a kit may include partially or whollydehydrated viral cultures of use in generating live, attenuated viruspopulations for vaccine production or other viral composition uses. Itis also contemplated that a kit may include one or more growth inducingcompositions disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments herein. Theembodiments may be better understood by reference to one or more ofthese drawings alone or in combination with the detailed description ofspecific embodiments presented.

FIG. 1 represents an exemplary graph of pluronic effects on viral growthafter cellular infection and introduction of a control agent orcopolymer composition during viral adsorption.

FIG. 2 represents an exemplary graph illustrating growth of viralcultures in the presence of a copolymer during viral adsorption and/orgrowth.

FIG. 3 represents an exemplary graph illustrating growth of viralcultures in the presence of increasing amounts of a copolymer-containingcomposition during viral adsorption and growth.

FIG. 4 represents an exemplary graph illustrating growth of viralcultures in the presence or absence of a specific copolymer added duringviral adsorption.

FIG. 5 represents an exemplary table illustrating change in plague sizeof exemplary viral cultures in the presence or absence of variousconcentrations of copolymer.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, vessel can include, but is not limited to, test tube,mini- or microfuge tube, plate, tissue culture flask, cell factory,channel, vial, microtiter plate or container.

As used herein the specification, “subject” or “subjects” may includebut are not limited mammals such as humans or mammals, domesticated orwild, for example dogs, cats, ferrets, rabbits, pigs, horses, cattle, orzoo animals.

As used herein, “about” can mean plus or minus ten percent.

As used herein, “high molecular weight surfactants” can mean a surfaceactive, amphiphilic molecule greater than 1500 molecular weight.

As used herein, “EO-PO block copolymer” can mean a copolymer consistingof blocks of poly(ethylene oxide) and poly(propylene) oxide. Inaddition, as used herein, “Pluronic” can mean EO-PO block copolymers inthe EOx-POy-EOx. This configuration of EO-PO block copolymer is alsoreferred to as “Poloxamer” or “Synperonic”.

As used herein, “attenuated virus” can mean a virus that demonstratesreduced or no clinical signs of viral-related disease when administeredto a subject such as a mammal (e.g. a human or an animal).

As used herein, “accelerate” can mean decreasing the lag time beforevirus production begins or increasing the rate of viral production suchthat higher concentrations of virus are produced in a shorter amount oftime or such that plaque size is increased in some embodiments relativeto a control.

As used herein, “killed virus vaccine” can mean a vaccine prepared byinactivating a virus by any of a number of physical or chemical meansknown in the art.

DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the details outlinedherein, but rather that concentrations, times and other details may bemodified through routine experimentation. In some cases, well-knownmethods or components have not been included in the description.

Embodiments herein concern using various compositions to enhance growthrate or reduce lag time of viral growth in a culture. In accordance withthese embodiments, compositions can include copolymer agents.Embodiments of this application generally relate to methods,compositions and uses for inducing and accelerating viral growth. Incertain embodiments, this application relates generally to methods,compositions and uses of copolymer compositions for accelerating viralgrowth and/or increasing viral plaque size. In other embodiments,methods, compositions and uses of copolymer compositions foraccelerating flaviviral growth, reducing lag in growth and/or increasingplaque size. Certain copolymer compositions contemplated herein include,but are not limited to, Pluronic F127, Pluronic F68, Pluronic P85,Pluronic P123, other EO-PO block copolymers of greater than 3,000-4,000MW or combinations thereof.

Copolymers

In certain embodiments, compositions can include copolymers, forexample, pluronic F127. Pluronic F127 (also referred to herein as F127)is a non-ionic polyoxyethylene-poloxypropylene copolymer. Pluronic blockcopolymers are known under their non-proprietary name as poloxamers.They were initially developed for use as surfactants. These compoundsconsist of hydrophilic ethylene oxide (EO) and hydrophobic propyleneoxide (PO) blocks. The EO-PO block copolymers can include blocks ofpolyethylene oxide (—CH2CH2O— designated EO) and polypropylene oxide(—CH2CHCH3O— designated PO). The PO block can be flanked by two EOblocks in an EOx-POy-EOx arrangement. Since the PO component ishydrophilic and the EO component is hydrophobic, overall hydrophilicity,molecular weight and the surfactant properties can be adjusted byvarying x and y in the EOx-POy-EOx block structure. According to themanufacturer, (e.g. BASF, Lutrol®F127) F127 can be used as a thickeningagent and co-emulsifier in creams and liquid emulsions.

F127 undergoes a process known as reverse thermogelation, as itundergoes a phase transition from liquid to a gel upon reachingphysiological temperatures. Higher temperatures promote the dehydrationof an alkylene oxide unit of the block polymer and this can result indecreased solubility. Specifically, at high concentrations (for example:about 10% w/v) certain types of the higher molecular weight EO-PO blockcopolymers will undergo reverse gelation, forming a gel as thetemperature increases. Additionally, when these block copolymers resideabove the critical micelle concentration (CMC), they self assemble intomicelles. In aqueous solutions, the EO-PO block copolymers willself-assemble into micelles with a PO core and a corona of hydrophilicEO groups. In certain studies, EO-PO block copolymer formulations havebeen investigated as potential drug delivery agents for a variety ofhydrophobic drugs and for protein, DNA or inactivated vaccines.

The mechanism of activity of these pluronic block copolymers iscurrently unknown. Although, Pluronic F127 has been studied as asustained release component of a vaccine delivery system in combinationwith chitosan. Vaccination of mice with Tetanus toxoid containing F127increased the antibody response in intranasally delivered andsystemically delivered tetanus antigens. In certain methods, pluronicshave been shown to induce changes in the microviscosity and fluidity ofcell membranes, which may contribute to its versatility.

Pluronic F127 has been used in a variety of human pharmaceuticalapplications including dental, oral and laxative pharmaceuticals.Vaccine formulations have also used surfactants as stabilizers toprevent material loss. Studies of DNA vaccine delivery with certainconcentrations of F127 (0.01% w/v) have shown increased drug delivery,possibly by potentiating cellular uptake and recruitment of maturedendritic cells. Gel formation at body temperatures permits use of theEO-PO block copolymer gels to act as a drug depot in vaccine and drugdelivery applications.

Certain compositions disclosed herein can include copolymers eitheralone or in combination with other agents or compounds. In addition,compositions disclosed herein may include a media composition having oneor more copolymer agent(s) added to the media in addition to other mediasupplements. Medias of use in compositions disclosed herein may includeany media known in the art known to grow viral organisms contemplatedherein or a media specific for a particular viral organism. Otherembodiments herein concern combinations of excipients that greatlyenhance the growth of live viruses (e.g. attenuated viruses). Yet othercompositions and methods herein are directed to reducing the lag timerelated to growth of viral organisms. Some embodiments concernmodulating plague size of viral organisms. Copolymers of use hereininclude, but are not limited to, Pluronic F127, Pluronic F68, PluronicP85, Pluronic P123, other EO-PO block copolymers of greater than3,000-4,000 MW or combinations thereof.

Compositions contemplated herein may be used alone or in combinationwith media before, during, and/or after viral cultures have beenintroduced to host culture cell media of compositions disclosed hereinmay be liquid, solid or semi-solid liquid. In certain embodiments,supplementary compositions may be added during entire viral growthperiods in order to monitor, adjust or stimulate viral growth processes.In other embodiments, one or more supplementary copolymer compositionsmay be added to reduce lag time, accelerate viral growth and/or increaseviral plaque size. Compositions contemplated herein may be used alone,in combination with other supplements (e.g. vitamins, metal ions andamino acids), or as a media supplement when media is added to thecultures.

Other embodiments include stocks for culturing viral cultures such aslive attenuated virus including, but not limited to, Picornaviruses(e.g., polio virus, foot and mouth disease virus), Caliciviruses (e.g.,SARS virus, feline infectious peritonitis virus), Togaviruses (e.g.,sindbis virus, the equine encephalitis viruses, chikungunya virus,rubella virus, Ross River virus, bovine diarrhea virus, hog choleravirus), Flaviviruses (e.g., dengue virus, West Nile virus, yellow fevervirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus), Coronaviruses (e.g., human coronaviruses(common cold), swine gastroenteritis virus), Rhabdoviruses (e.g., rabiesvirus, vesicular stomatitis viruses), Filoviruses (e.g., Marburg virus,Ebola virus.), Paramyxoviruses (e.g., measles virus, canine distempervirus, mumps virus, parainfluenza viruses, respiratory syncytial virus,Newcastle disease virus, rinderpest virus), Orthomyxoviruses (e.g.,human influenza viruses, avian influenza viruses, equine influenzaviruses), Bunyaviruses (e.g., hantavirus, LaCrosse virus, Rift Valleyfever virus), Arenaviruses (e.g., Lassa virus, Machupo virus),Reoviruses (e.g., human reoviruses, human rotavirus), Birnaviruses(e.g., infectious bursal virus, fish pancreatic necrosis virus),Retroviruses (e.g., HIV 1, HIV 2, HTLV-1, HTLV-2, bovine leukemia virus,feline immunodeficiency virus, feline sarcoma virus, mouse mammary tumorvirus), Hepadnaviruses (e.g., hepatitis B virus.), Parvoviruses (humanparvovirus B, canine parvovirus, feline panleukopenia virus)Papovaviruses (e.g., human papillomaviruses, SV40, bovinepapillomaviruses), Adenoviruses (e.g., human adenovirus, canineadenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses(e.g., herpes simplex viruses, varicella-zoster virus, infectious bovinerhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), andPoxviruses (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, skunkpoxvirus, monkeypoxvirus, cowpox virus, musculum contagiosum virus).

In accordance with these embodiments, certain live attenuated virusesinclude, but are not limited to, live, attenuated flaviviruses. Someembodiments, directed to compositions, can include, but are not limitedto, one or more live, attenuated viruses, such as one or more live,attenuated flaviviruses grown in one or more copolymer compositionsalone or in combination with other agents. In accordance with theseembodiments, a flavivirus can include, but are not limited to, denguevirus, West Nile virus, yellow fever virus, Japanese encephalitis virus,St. Louis encephalitis virus, tick-borne encephalitis virus or otherknown flavivirus.

In other embodiments, compositions contemplated herein can increaseplaque size in reduced or similar time periods of growth, compared tocontrols not grown compositions disclosed herein, for use in assessingviral activity or tittering viral preparations. Alternatively,compositions contemplated herein can reduce lag time or accelerategrowth time for up to several days earlier than control viral culturesnot using compositions contemplated herein. In certain embodiments,predetermined viral titers may occur several hours, a half a day, 1 day,2 days, 3 days, 4 days or even up to 10 days earlier than viruspreparations grown in other media known in the art or supplementalcompositions furnished to cultures having no copolymer. Optimal viraltiter of some embodiments may be about 1×10⁶ pfu/mL to about 1×10⁸pfu/mL. In certain embodiments, a flaviviral titer may reachconcentrations of about 1×10⁷ pfu/mL in about 4 days in media containingF127, as compared to cultures grown in media without F127 which takesabout 6 days.

Some embodiments herein concern compositions and methods for modulatingtime for growth of a viral culture to reach a predeterminedconcentration. In accordance with these embodiments, time for growth maybe reduced by about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40% and more. In various embodiments, apredetermined viral culture density may be accomplished in about 80%, orabout 70%, or about 60% of time using compositions disclosed hereincompared to other compositions known in the art.

In certain embodiments, viral cultures contemplated for productionherein may be used in compositions including, but not limited to,partially or wholly dehydrated or hydrated vaccine formulations. Inother embodiments, viral cultures contemplated herein for production ofvaccine formulations can be cultured for reduced time and costs. Inaddition, production of these vaccine formulations can be reduced inlabor, time and costs, for example, in times when an epidemic oroutbreak of flaviviral-associated diseases occur and vaccineformulations are required in a short period of time.

In some embodiments, a live attenuated virus for use in a vaccinecomposition contemplated herein may include, but is not limited to, oneor more live, attenuated flavivirus vaccines, including but not limitedto, attenuated yellow fever viruses (such as 17D), attenuated Japaneseencephalitis viruses, (such as SA 14-14-2), attenuated dengue viruses(such as DEN-2/PDK-53 or DEN-4Δ30), attenuated chimeric West Nile virus,or recombinant chimeric flaviviruses. In certain embodiments, theflaviviral cultures of use in a vaccine composition can be grown inmedia compositions having one or more copolymer disclosed herein.

Other embodiments concern virus populations of use in formulations andmethods directed to vaccine formulations capable of reducing orpreventing onset of a medical condition caused by one or more of theflaviviruses contemplated herein. In accordance with these embodiments,medical conditions may include, but are not limited to, West Nileinfection, dengue fever, Japanese encephalitis, Kyasanur forest disease,Murray valley encephalitis, Alkhurma hemorrhagic fever, St. Louisencephalitis, tick-borne encephalitis, yellow fever and hepatitis Cvirus infection. Thus, production time for generating these formulationscan be reduced using compositions contemplated herein for increasinggrowth, reducing lag time and/or increasing plague size of viralpopulations used in formulations disclosed.

Other embodiments concern virus compositions of use in therapeuticapplications. Such uses may include, but are not limited to, genetherapy applications. Viruses used to deliver genes to cells in genetherapy applications include lentiviruses, adenoviruses,adeno-associated viruses, and herpesviruses. Other uses of viruscompositions may include, but are not limited to, cancer virus therapies(e.g., “oncolytic” viruses) or cancer immunotherapies.

It is contemplated herein that any media used for growth of cellcultures (e.g. host cells) may be of use herein. For example, commonlyused medias for cell cultures are contemplated. In accordance with theseembodiments, media may include, but are not limited to DMEM (Dulbecco'sModified Eagle Medium, high glucose, with L-glutamine, with pyridoxinehydrochloride, without sodium pyruvate containing 3.7 g sodiumbicarbonate per liter), MEM, BSS/YE-LAH, F-10 (Ham's), F-12, M-199,RPMI, Agars, LB Broth, and PBS- based medias. In addition, it iscontemplated that cells may be cultured by any means known in the art.For example, cells may be grown in confluent layers, as suspensions, inmultiple layers, in roller bottles, in wells or in tubes.

In certain embodiments, host cells can be used to culture virusesdisclosed herein. Any cell known to host viruses disclosed herein iscontemplated. Some host cells of use for growing viruses disclosedherein include, but are not limited to, Vero (African green monkey Verocells), LLC-MK₂ cells, C6/36 mosquito cells or other cells known in theart.

Some embodiments of the present invention report compositions having oneor more high molecular weight surfactants or copolymer compounds of usein methods for culturing various viral cultures where some compositionsdisclosed herein are capable of modulating various aspects of viralgrowth (e.g. larger plaque size, reduced lag phase) by about 10%, byabout 15%, by about 20%, by about 25%, by about 30%, by about 35%, byabout 40%, by about 45%, by about 50% or more, compared to compositionsnot having a copolymer composition.

Kits

Further embodiments concerns kits of use for methods and compositionsdescribed herein. Compositions including, but not limited to, copolymercompositions and live virus formulations may be provided in a kit. Kitscan also include, but are not limited to, a suitable container,copolymer compositions, live virus compositions detailed herein andoptionally, one or more additional agents such as other anti-viralagents, anti-fungal agents or anti-bacterial agents for example, tomodulate growth of undesireable species.

The kits may further include a suitably aliquoted copolymer compositionof use for viral cultures. In addition, compositions herein may bepartially or wholly dehydrated or aqueous viral cultures and/or hostcells for propagating the viruses as well as liquid or partially orwholly dehydrated medias. Kits contemplated herein may be stored at roomtemperatures, frozen or at refrigerated temperatures as disclosed hereindepending on the particular formulations and components.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich a composition may be placed, and preferably, suitably aliquoted.Where an additional component is provided, the kit will also generallycontain one or more additional containers into which this agent orcomponent may be placed. Kits herein will also typically include a meansfor containing the agent, composition and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

The following examples are included to demonstrate certain embodimentspresented herein. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered to function well in the practices disclosedherein, and thus can be considered for its practice. However, those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope herein.

Examples Example 1

In one exemplary method, represented in FIG. 1, pluronic effects onflavivirus growth were examined in one exemplary cell line, Vero cells(African green monkey Vero cells). Vero cells were grown to confluencyfor example, in T-75 cm2 flasks 2 days prior to infection withflavivirus (as indicated) at an MOI of 0.001. Virus adsorption for 180minutes was assessed in 2 mL PBS in the presence or absence of pluronic(P123 or F127). Control samples contained viral adsorption in PBSwithout a copolymer. Growth media (18 mL serum-free DMEM) was addedafter adsorption. Aliquots were taken daily, and titrated on Vero cellmonolayers. Viral titers were measured as illustrated in FIG. 1.

In another example, illustrated in FIG. 2, growth of the chimericflavivirus DEN-2/4 in Vero cells, without or with varying concentrationsof copolymer, Pluoronic F127 was examined. Vero cells were grown toconfluency for example, in T-75 cm2 flasks 2 days prior to infectionwith DEN-2/4 at an MOI of 0.001. Virus was adsorbed for 120 minutes in 2mL DMEM with or without F127. Viral inoculums were rinsed from the cellmonolayer with PBS followed by addition of 25 mL indicated growth mediumcontaining 5% FBS with or without F127 and grown for 14 days. Aliquotswere taken daily, and titrated on vero cell monolayers. Viral titerswere measured as illustrated in FIG. 2.

Example 2

In another exemplary method, growth of DEN 2/4 (Dengue 2/4) chimera inVero cells containing increasing amounts of F127 during adsorption andgrowth were examined (see for example, FIG. 3). A confluent monolayer ofVero cells grown in T75 cm2 flasks in 25 mL DMEM medium containing 10%FBS and control or increasing concentrations of F127. Exemplary F127concentrations used in this experiment included 0.063%, 0.125%, 0.25%,0.5%, 1.0% and 2.0% F127. The cells were infected with DEN2/4 at MOI=0.001. Parameters included adsorption for 1.5 hours in lmL DMEM/F127.Aliquots were taken every other day, and titrated on Vero cellmonolayers. Viral titers were measured as illustrated in FIG. 3.

In addition, another experiment analyzed effects of the copolymer F127during viral adsorption of the chimeric flavivirus DEN2/1. In thisexample, DEN2/1 was adsorbed onto a confluent flask of Vero cells at anMOI of 0.001 for 90 minutes. Adsorption was performed in 1 mL of growthmedia (BA-1) with or without 1% F127. After viral adsorption, 20 mL DMEMcontaining 2% FBS and no F127 was added to the cultures. Aliquots weretaken every other day and titrated on Vero cell monolayers. Viral titerscan be measured as illustrated in FIG. 4.

Example 3

Another exemplary method analyzed whether or not plaque size increasedin the presence of an exemplary copolymer, F127. FIG. 5 represents anexemplary table, Table 1. This experiment demonstrated that flavivirusplaque size increases in the presence of increasing concentrations ofpluronic F127 during growth. Here, confluent monolayer of Vero cellswere grown in T75 cm2 flask in 20 mL DMEM2% FBS medium in the presenceor absence of F127 where the Vero cells were infected with DEN2/4 atMOI=0.001. Adsorption was 1.5 hours and 1 mL DMEM in the presence (0.1%or 1.0%) or absence of F127. Plaques (e.g. 8) were visualized on a lightbox, and their diameter was measured. Table 1 represents results of oneway ANOVA of plaque size (mm) differences in DENVax 2/4 growth in theabsence of F127 or presence of increasing concentrations of F127.

Materials and Methods:

It is contemplated that any method known in the art can be used for anycomposition, method and/or uses described herein. In certainembodiments, it is contemplated that certain methods will be more suitedfor viral growth, for example materials and methods of use forflaviviral growth, than other methods. In other embodiments, it iscontemplated that certain methods will be more suited for the growth ofDengue, than suited for other flaviviruses. The following provides abrief description as to the methods to grow a high-titer chimeric Denguevaccine or any live-attenuated flavivirus in the presence of F127. UsingT-75 cm2 flask, for example, Vero cells are seeded 2 days prior to viralinfection/adsorption at a density of 5×10̂6 cells per flask. This viralgrowth can be “scaled up” to include tissue culturing vessels rangingfrom T-25 cm2 to 10-stack cell factories. Virus is adsorbed onto aconfluent monolayer of Vero cells 2 days after cell seeding in lmL DMEMcontaining F127 (0.1%). The culture vessels are incubated for 1.5 hoursat 37° C. with rocking of the vessel every 10 minutes. After viraladsorption, the cell monolayers are washed three times with 10 mL PBS.Growth medium (10 mL DMEM pH=7.2, containing 3.7 g/L NaHCO₃ and 0.1%F127 with no FBS) is then added to the monolayers, and incubated with orwithout aeration at 37° C. for 4 days. On day 4 of viral growth, thegrowth medium is replaced, as done after viral adsorption. Starting atday 6, and until day 12, the infectious medium is completely removedfrom the growth chamber and centrifuged for clarification. This viralgrowth is stabilized, and stored at −80° C. until its titer can beexamined by plaque assay on Vero cell monolayers. After daily harvests,the growth medium is replaced on the tissue culture vessels as done onday 4. Daily harvests continue until day 12. Daily harvests areindividually titered on Vero cells, and high-titer harvests can bepooled to obtain a homogeneous sample. Often, the first day harvest (day6) is not included, to avoid high levels of host-cell (Vero) DNA.

TABLE 1 Example of DMEM - F12: F-12 Nutrient Mixture (Ham), powder(21700) with L-glutamine Additives per 10 L: 11.76 g Sodium Bicarbonate,100 ml Penicillin Streptomycin Mole. Conc. Molarity COMPONENTS Weight(mg/L) (mm) INORGANIC SALTS: Calcium chloride (Anhydrous) 111 33.220.299 Cupric sulfate (CuSO4—5H2O) 250 0.0025 0.00001 Ferric sulfate(FeSO4—7H2O) 278 0.834 0.003 Potassium chloride (KCl) 75 223.60 2.98Magnesium chloride (Anhydrous) 95 57.22 0.60 Sodium chloride (NaCl) 587599.00 131.00 Sodium bicarbonate (NaHCO3) 84 1176.00 14.00 Sodiumphosphate, dibas 142 142.00 1.00 (Anhydrous) Zinc sulfate (ZnSO4—7H2O)288 0.863 0.003 OTHER COMPOUNDS: D-Glucose 180 1802.00 1.00 HypoxanthineNa 159 4.77 0.03 Linoleic Acid 280 0.084 0.0003 Lipoic Acid 206 0.210.000971 Phenol red 398 1.20 0.003 Putrescine-2HCl 161 0.161 0.001Sodium Pyruvate 110 110.00 1.00 Thymidine 242 0.70 0.003 AMINO ACIDS:L-Alanine 89 8.90 0.100 L-Arginine hydrochloride 211 211.00 1.00L-Asparagine-H2O 150 15.01 0.100 L-Aspartic acid 133 13.30 0.100L-Cysteine-HCl—H2O 176 35.12 0.200 L-Glutamic acid 147 14.70 0.100L-Glutamine 146 146.00 1.00 Glycine 75 7.50 0.100 L-Histidine-HCl—H2O210 21.00 0.0998 L-Isoleucine 131 4.00 0.030 L-Leucine 131 13.10 0.100L-Lysine hydrochloride 183 36.50 0.199 L-Methionine 149 4.50 0.030L-Phenylalanine 165 5.00 0.030 L-Proline 115 34.50 0.300 L-Serine 10510.50 0.100 L-Threonine 119 11.90 0.100 L-Tryptophan 204 2.04 0.010L-Tyrosine 2Na 2H2O 225 7.81 0.03 L-Valine 117 11.70 0.100 VITAMINS:Biotin 244 0.0073 0.00003 D-Calcium pantothenate 477 0.50 0.001 Cholinechloride 140 14.00 0.0997 Folic acid 441 1.30 0.0029 i-Inositol 18018.00 0.100 Niacinamide 122 0.036 0.0003 Pyridoxine hydrochloride 2060.06 0.0003 Riboflavin 376 0.037 0.000101 Thiamine hydrochloride 3370.30 0.001 Vitamin B12 1,355 1.40 0.001 Adjust the pH to 7.2

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

1. A composition for growing viral cultures comprising: one or more highmolecular weight surfactants or copolymers; and optionally, a media,wherein the high molecular weight surfactants or copolymer accelerategrowth of viral cultures.
 2. The composition of claim 1, wherein thehigh molecular weight surfactants or copolymers comprise Pluronic F127,Pluronic F68, Pluronic P85, Pluronic P123, other EO-PO block copolymersof greater than 3,000-4,000 MW or a combination thereof.
 3. Thecomposition of claim 1, wherein the viral cultures are selected from thegroup consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus,Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus,Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus,Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, andPoxvirus.
 4. The composition of claim 1, wherein the viral cultures areFlavivirus cultures.
 5. The composition of claim 1, wherein the viralcultures are Poxvirus cultures.
 6. The composition of claim 1, whereinthe high molecular weight surfactants comprise EO-PO block copolymerPluronic F127, Pluronic P123 or a combination thereof.
 7. Thecomposition of claim 1, wherein at least one of the high molecularweight surfactants has a molecular weight of 1500 or greater.
 8. Thecomposition of claim 1, wherein at least one of the high molecularweight surfactants further comprises one or more copolymers and whereinthe molecular weight of the at least one high molecular weightsurfactant agent is 3000 or greater.
 9. The composition of claim 1,wherein at least one of the high molecular weight surfactantsconcentration is about 0.001% to about 3.0%.
 10. The composition ofclaim 1, wherein at least one of the high molecular weight surfactantscomprises Pluronic F127 and the media comprises Dulbecco's ModifiedEagle Medium (DMEM).
 11. A method for increasing viral growth ratecomprising, administering to a host cell culture infected with a virus,a composition comprising one or more high molecular weight surfactantsor copolymers wherein the composition increases viral growth rate.
 12. Amethod for increasing viral growth rate comprising, administering acomposition comprising one or more high molecular weight surfactants orcopolymers to a host cell culture, before, during, or after viralinfection of the host cell culture.
 13. A method for increasing plaquesize of a viral culture comprising, administering to a host cell cultureinfected with a virus, a composition comprising one or more highmolecular weight surfactants or copolymers wherein the compositionincreases viral plaque size compared to a control viral culture withoutadministering one or more high molecular weight surfactants orcopolymers.
 14. A method for reducing growth lag time of a viral culturecomprising, administering to a host cell culture infected with a virus,a composition comprising one or more high molecular weight surfactantsor copolymers wherein the composition reduces lag time of the viralcultures compared to a control viral culture without administering oneor more high molecular weight surfactants or copolymers.
 15. The methodof claim 14, wherein the viral cultures are selected from Flaviviralcultures.
 16. The method of claim 14, wherein the viral culturescomprise viral cultures for generating live, attenuated viral vaccines.17. The method of claim 14, wherein the reduction in lag time comprisesat least a 10 percent reduction in lag time compared to viral cultureswithout one or more high molecular weight surfactants or copolymers. 18.The method of claim 14, wherein at least one high molecular weightsurfactants comprise Pluronic F127, Pluronic F68, Pluronic P85, PluronicP123, other EO-PO block copolymers of greater than 3,000-4,000 MW or acombination thereof.
 19. A kit for culturing viruses comprising; atleast one container; a composition comprising one or more high molecularweight surfactants; and optionally, one or more viral cultures.
 20. Thekit of claim 19, wherein at least one of the high molecular weightsurfactants comprise Pluronic F127, Pluronic F68, Pluronic P85, PluronicP123, other EO-PO block copolymers of greater than 3,000-4,000 MW or acombination thereof.
 21. The kit of claim 19, wherein the EO-PO blockcopolymer Pluronic F127 concentration is from 0.001% to about 3.0%. 22.The kit of claim 19, wherein the viral culture comprises one or moreFlaviviruses.
 23. The kit of claim 19, further comprising a media. 24.The kit of claim 19, further comprising a host cell stock culture.